Prodrug of 1,1&#39;-(1,6-dioxo-1,6-hexanediyl)bis-d-proline

ABSTRACT

The present invention relates to the compound (2R,2′R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl) 1,1′-adipoylbis(pyrrolidine-2-carboxylate), pharmaceutical compositions comprising the same and the use of the same for treatment of diseases or disorders wherein depletion of serum amyloid P component (SAP) would be beneficial, including amyloidosis, Alzheimer&#39;s disease, type 2 diabetes mellitus and osteoarthritis.

FIELD OF THE INVENTION

The present invention relates to the novel compound(2R,2′R)-Bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl)1,1′-adipoylbis(pyrrolidine-2-carboxylate), pharmaceutical compositionscomprising the same and the use of the same for treatment of diseases ordisorders wherein depletion of serum amyloid P component (SAP) would bebeneficial.

BACKGROUND TO THE INVENTION

Serum amyloid P component (SAP) is a normal, structurally invariant,soluble, non-fibrillar, constitutive plasma glycoprotein, mass 127,310Da, produced exclusively by the liver. It is composed of 5 identical25,462 Da protomers non-covalently associated with cyclic pentamericsymmetry in a disc like configuration. Each subunit, composed of aflattened β-jellyroll, with tightly tethered loops joining theβ-strands, contains a single calcium dependent ligand binding site onthe planar B (binding) face of the intact pentamer. SAP binds to alltypes of amyloid fibrils with the high avidity conferred by multivalentinteractions. This strictly calcium dependent interaction is responsiblefor the universal presence of human SAP in all human amyloid deposits ofall types, and hence the name of the protein, where P stands for plasma,the source of this component of amyloid. In addition to its capacity forspecific calcium dependent binding to particular ligands, a key propertyof human SAP is that the protein itself is intrinsically resistant toproteolysis. Its avid binding to amyloid fibrils is mutuallystabilising, strongly protecting the fibrils against proteolysis andphagocytic degradation in vitro¹ and significantly contributing topersistence of amyloid in vivo². These observations underlie thevalidation of SAP as a therapeutic target in amyloidosis (M B Pepys & TL Blundell, U.S. Pat. No. 6,126,918, 3 Oct. 2000). Furthermore, bindingof SAP to nascent amyloid fibrils strongly promotes amyloidfibrillogenesis³⁻⁵. European patent application EP 0915088 disclosescompounds which are competitive inhibitors of binding of SAP to amyloidfibrils, as well as methods for their manufacture. One of the compoundsdisclosed therein is(R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid (CPHPC).

Administration of these palindromic bivalent ligands for SAP causes therapid and almost complete depletion of SAP from the circulation for aslong as the compounds are administered^(6,7), as described inWO2003/013508, U.S. Pat. No. 7,045,499; U.S. Pat. No. 7,691,897; andU.S. Pat. No. 8,173,694. This treatment also reduces the amount of SAPassociated with the amyloid deposits but does not remove all the SAP⁷.

Amyloid is an abnormal, insoluble, extracellular deposit, composedpredominantly of characteristic protein fibrils⁸ together with abundantproteoglycans and glycosaminoglycans. About 25 different, unrelated,natively soluble, globular proteins form the amyloid fibrils which causethe different types of systemic amyloidosis but all amyloid fibrils havevery similar morphology and the same cross-β core structure. Thisstructure binds the ordered arrays of Congo red dye molecules whichcreate pathognomonic red-green birefringence in strong cross polarisedlight: the gold standard diagnostic criterion for amyloid. Certainsoluble, non-fibrillar plasma proteins may also be present in amyloiddeposits but only one, serum amyloid P component (SAP), is universal inall human amyloid deposits, due to its avid, specific, calcium dependentbinding to all types of amyloid fibrils.

Amyloid deposits disrupt the structure and function of affected tissuesand organs, causing the serious disease, amyloidosis. Systemicamyloidosis is a rare, fatal condition caused by amyloid deposits whichmay be present in connective tissue and blood vessel walls throughoutthe body, as well as the parenchyma of the major organs, but never inbrain substance itself. In local amyloidosis, the amyloid deposits areconfined to a single anatomical site or a single organ/tissue system.Cerebral amyloid angiopathy, with amyloid deposition confined to thewalls of cerebral blood vessels, is the most common and important formof local amyloidosis. It is responsible for a substantial proportion ofintracerebral haemorrhages in both demented Alzheimer's disease patientsand non-demented individuals, and is thus an important cause of dementiain its own right.

Treatment of systemic amyloidosis patients with CPHPC produced almostcomplete depletion of circulating SAP for as long as the drug wasadministered but did not remove all the SAP bound to the amyloiddeposits.⁷ The patients remained clinically stable while being treated,with no new amyloid accumulation, and their organ function wasmaintained but there was no regression of amyloid, probably due to thefailure of complete removal of amyloid bound SAP. Since the amyloiddeposits in the tissues are the direct cause of disease, it is highlydesirable that they should be eliminated. This important unmet medicalneed led to the invention of a new approach to treatment of amyloidosisin which SAP bound to amyloid deposits is used as a target foranti-human SAP antibodies. Such antibodies could not be safely oreffectively administered to patients with normal circulatingconcentrations of SAP since the antibodies would bind to the SAP in theplasma, forming tissue damaging immune complexes, and the antibodieswould be consumed in this process making them unavailable for binding toSAP in amyloid. However prior administration of CPHPC depletes SAP fromthe circulation, so that anti-SAP antibodies can be safely infused andremain available to bind to residual SAP left in the amyloid deposits.Binding of the antibodies to the amyloid-associated SAP activates thecomplement system and engages macrophages to phagocytose and destroy theamyloid deposits leading to clinical benefit.

International patent application WO2009/000926 discloses the use ofcompounds which deplete SAP from the circulation co-administered with anantibody specific for SAP for potential treatment of amyloidosis.

International patent application WO2009/155962 discloses mousemonoclonal antibody Abp1 and provides binding and efficacy data forvarious mouse monoclonal antibodies which may be co-administered withcompounds which deplete SAP from the circulation for potential use inthe treatment of amyloidosis.

International patent application WO2011/107480 discloses antigen bindingproteins, in particular humanised antibodies, specific for SAP and theirpotential use in the treatment of diseases associated with amyloiddeposition.

In addition to the rare clinical condition of amyloidosis, which isunequivocally directly caused by extracellular amyloid depositiondisrupting tissue structure and function, amyloid deposits are alsopresent in two very common and important diseases: Alzheimer's diseaseand type 2 diabetes. In these latter diseases, the amyloid deposits aremicroscopic, are confined to the brain and islets of Langerhansrespectively, and it is not known whether or how they may contribute topathogenesis of neurodegeneration and diabetes respectively. Alzheimer'sdisease and type 2 diabetes thus cannot be classified as forms ofamyloidosis but rather should be considered as amyloid associateddiseases. Nevertheless, amyloid deposits are always present in them andthe deposits also always contain SAP¹⁵⁻¹⁹. The brain in Alzheimer'sdisease also contains another type of abnormal insoluble fibrillarprotein aggregate, known as neurofibrillary tangles, and SAP bindsavidly to these, as it does to amyloid.¹⁵⁻¹⁹ Neurofibrillary tanglesbearing SAP, but not amyloid, are present in the brain in other types ofdementia, including the frontotemporal dementias

In addition to, and quite independent of, its role in amyloidosis, humanSAP binds to and enters cerebral neurones and causes neuronal apoptosisin vitro and in vivo⁹⁻¹³. It has been shown that unique, pharmaceuticalgrade pure human SAP″ disrupts synaptic transmission, causing abnormalpaired pulse ratio and long term potentiation in organotypic rodentbrain slices in vitro.

The cerebral neurotoxicity of human SAP is therefore likely tocontribute to neurodegeneration in humans^(9-12,20). The fact that mostof the common risk factors for dementia increase brain exposure to SAPis consistent with this concept. Thus age, a key risk factor, isassociated with prolonged exposure of the ageing brain to normal SAPconcentrations, whilst the major risk factors of non-penetrating headtrauma and cerebral haemorrhage cause blood to enter the brain, sharplyincreasing cerebral SAP content. In Alzheimer's disease, brain contentof SAP is abnormally high due to its binding to amyloid deposits andneurofibrillary tangles¹⁵⁻¹⁹. This is also likely to be the case inother dementias with neurofibrillary tangles but not amyloid deposits.Importantly, higher brain SAP content is reported in dementedAlzheimer's disease patients than in elderly subjects who werecognitively intact at death, either with or without co-existing plaquesand tangles at autopsy²⁰. The significant positive correlation betweencerebral SAP content and dementia²⁰ is consistent with a causative rolefor SAP.

The quantities of SAP in cerebrospinal fluid and bound to cerebralamyloid deposits and neurofibrillary tangles are dramatically lower thanin systemic extracellular fluid and on systemic amyloid depositsrespectively. Depletion of plasma SAP by CPHPC, from the normal 20-50mg/L to <0.1 mg/L, reduces the CSF SAP concentration from 2-30 μg/L to<0.1 μg/L in patients with Alzheimer's disease²¹. Human SAP is producedonly by the liver and reaches the brain via the blood²². CPHPC treatmentthus removes virtually all SAP from the cerebrospinal fluid and, sinceSAP binding is fully reversible, will also remove it from the cerebralamyloid deposits and neurofibrillary tangles. Furthermore in Alzheimer'sdisease patients, CPHPC enters the cerebrospinal fluid²¹ where it canalso block binding of any free SAP to amyloid fibrils, toneurofibrillary tangles and cerebral neurones. All amyloid fibril typescan be degraded by proteases and phagocytic cells in vitro¹, andsystemic amyloid deposits spontaneously regress slowly in vivo when theabundance of their respective fibril precursor proteins is sufficientlyreduced⁸. Mechanisms for amyloid clearance thus do operate in vivo.Confirmation that human SAP is itself neurocytotoxic⁹⁻¹³, independent ofits binding to amyloid, demonstrates the potential, additional, directbenefit of SAP depletion.

All plasma proteins enter diseased or damaged joints in vivo but inpatients with various different arthropathies, uptake of radiolabelledSAP into some joints that did not have clinically detectable effusionswas observed. Furthermore the concentration of SAP in synovial effusionfluids was substantially below that predicted from the molecular size ofSAP, demonstrating that the SAP visualised scintigraphically was notfree in solution but was actually bound to solid structures within thejoint. Synovium, articular cartilage and/or joint capsules of elderlyindividuals often contain microscopic amyloid deposits, associated withage rather than extent or severity of osteoarthritis, and these depositscould explain the localisation of SAP. SAP also binds avidly, in vivo aswell as in vitro, to exposed DNA and chromatin, and to apoptotic cells.Increased cell death in osteoarthritic joints may thus provide ligandsfor SAP deposition. WO2004/108131 discloses the treatment of patientswith osteoarthritis by injection of CPHPC((R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid) resulting in the alleviation of osteoarthritis symptoms.

Human SAP binds avidly to all forms of free DNA and also to chromatin,both in vitro and in vivo. Indeed SAP is the only normal human plasmaprotein which binds specifically in a calcium dependent interaction withDNA^(23,24). In contrast, SAP from some other species, including mouseand horse, binds weakly if at all to DNA, and dogs and rabbits do noteven have the SAP gene and thus produce no SAP. DNA vaccination, inwhich immunisation is achieved by injection of DNA coding for theimmunogen rather by injection of the immunogen itself, has beenextensively investigated as a highly desirable approach to induction ofprotective immunity against infections and as a potentialimmunotherapeutic intervention in cancer. However, although DNAvaccination is effective in mice. dogs, rabbits and horses, it hasconsistently failed in humans and also in cows, which like humans haveSAP which binds avidly to DNA. In the species in which DNA vaccinationworks, SAP either does not bind to DNA or is absent. Furthermore,experiments in mice with transgenic expression of human SAP and usingCPHPC to deplete it, confirm that the presence of human SAP blocksefficacy of DNA vaccination.^(25,26)

SAP binds to some bacterial species but not to others. For thosepathogenic bacteria to which SAP binds, the SAP has a powerfulanti-opsonic effect in vitro and in vivo, reducing phagocytosis andkilling of the organisms and thus protecting them from the host's innateimmune system²⁷. This effect, which promotes infectivity and virulence,is abrogated by administration of CPHPC to inhibit binding of SAP to thebacteria²⁷. SAP is also present bound to the surface of the fungal cellsin the tissues of patients suffering from invasive candidiasis²⁸. Thisbinding reflects the presence of amyloid fibrils, formed from fungalproteins, on the surface of the pathogenic organism.²⁸

CPHPC is pharmacologically effective but it has very low and variableoral bioavailability of ˜3-5% and therefore only parenteraladministration by intravenous infusion or subcutaneous injection isoptimal to achieve the desired SAP depletion. However most of theexisting and potential indications for therapeutic SAP depletion requirelong term administration. Long term intravenous administration is notpractical. Although long term subcutaneous administration is feasible,the injections may cause stinging discomfort and this is not toleratedby some patients.⁷

WO2003/051836 discloses D-prolines pro-drugs useful for the treatment ofdiseases where SAP depletion has a beneficial effect. The 25 Examplesdisclosed therein were predominantly obtained as oils; only 5 of themwere solid. In proceedings at the European Patent Office, a divisionalapplication to the European equivalent of WO2003/051836 was filed withclaims directed to(R)-1-(6-{(R)-2-[1-(2,2-dimethyl-propionyloxy)-ethoxycarbonyl]-pyrrolidin-1-yl}-6-oxo-hexanoyl)-pyrrolidine-2-carboxylicacid 1-(2,2-dimethyl-propionyloxy)-ethyl ester. At the time of filing,the GlaxoSmithKline group of companies (Glaxo Group Limited) has aLicence and Research Collaboration Agreement with Pentraxin TherapeuticsLimited including a licence to EP 0915088 and WO2003/051836, andcorresponding applications thereof.

There is therefore a need for a compound which is capable of generatingCPHPC in quantities capable of depleting SAP efficiently following oraladministration, whilst possessing physicochemical properties suitablefor pharmaceutical development.

SUMMARY OF THE INVENTION

It has surprisingly been found that the compound(2R,2′R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl)1,1′-adipoylbis(pyrrolidine-2-carboxylate) according to Formula (I)possesses physicochemical properties suitable for pharmaceuticaldevelopment and is capable of generating CPHPC in quantities capable ofdepleting SAP efficiently following oral administration.

Accordingly, in a first aspect, the present invention provides acompound(2R,2′R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl)1,1′-adipoylbis(pyrrolidine-2-carboxylate) according to Formula (I):

The compound of Formula (I) is referred to hereinafter as “the compoundof the invention”.

The compound of the invention exhibits good physiochemical propertiesand is capable of generating CPHPC in quantities capable of depletingSAP efficiently following oral administration.

The compound of Formula (I) has good pH solution stability andintestinal microsome stability, and low liver microsome stability,readily generating CPHPC, suggesting the compound of Formula (I) willreadily generate circulating levels of CPHPC on oral dosing in humans.Also, it does not show any interaction with cytochrome p450, suggestingthe compound of Formula (I) will not show CYP450 mediated drug-druginteractions (DDIs). Additionally, the compound of Formula (I) is highlycrystalline, which is advantageous with respect to formulation of theactive substance and manufacture of the pharmaceutical product.

The use of a compound with this profile may result in advantages in thetreatment of diseases or disorders wherein depletion of SAP (serumamyloid P component) would be beneficial, for example in amyloidosis(including systemic amyloidosis and local amyloidosis), Alzheimer'sdisease, type II diabetes, osteoarthritis, bacterial infection, invasivecandiasis and other fungal infections, and in conjunction withadministration of DNA vaccines.

In a further aspect the invention provides a pharmaceutical composition,which comprises a therapeutically effective amount of a compound ofFormula (I) and optionally one or more pharmaceutically acceptablecarriers and/or excipients.

In a further aspect there is provided a compound of Formula (I) or oneor more pharmaceutical compositions herein described for the depletionof circulating SAP in a subject.

In a further aspect there is provided the compound of Formula (I) or oneor more pharmaceutical compositions herein described, for use in thetreatment of diseases wherein SAP depletion would be beneficial.

In a further aspect there is provided a method of treatment of a diseaseor disorder wherein SAP depletion would be beneficial.

In a further aspect there is provided the use of a compound of Formula(I) or one or more pharmaceutical compositions herein described, in themanufacture of a medicament for use in the treatment of diseases whereinSAP depletion would be beneficial.

In a further aspect there is provided a kit of parts comprising one ormore dosage forms of an anti-SAP antibody (in particular an antibody asdisclosed in WO2011/107480) and one or more dosage forms of the compoundof Formula (I).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an X-ray powder diffraction pattern (XRPD) of thecrystalline solid form of the compound of Formula (I).

FIG. 2 shows in vitro physical hydrolysis of the compound of Formula (I)in phosphate buffered saline at pH 6, 7, 7.5 and 8.

FIG. 3 shows in vitro Intestinal Microsomal Stability of the compound ofFormula (I) in human microsomes.

FIG. 4 shows in vitro Blood stability of the compound of Formula (I) inhuman blood.

FIG. 5 shows in vitro Liver microsomal stability of the compound ofFormula (I) in human liver microsomes.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following terms are intended to have the meanings presented herewithbelow and are useful in understanding the description and the scope ofthe present invention.

“Pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal government of the United States ofAmerica or the corresponding agency in countries other than the UnitedStates of America (such as the EMA, the European Medicines Agency), orthat is listed in the United States Pharmacopeia or EuropeanPharmacopoeia (Ph. Eur.).

“Therapeutically effective amount” means the amount of a compound that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease.

The term “antibody” is used in the broadest sense to refer to moleculeswith an immunoglobulin-like domain and includes monoclonal, recombinant,polyclonal, chimeric, humanised, human, bispecific and heteroconjugateantibodies; and antigen binding fragments. An antibody according to thepresent invention activates the human complement system and/or resultsin regression of amyloid deposits.

It will be appreciated that reference to “treatment” includes acutetreatment or prophylaxis as well as the alleviation of establishedsymptoms and/or retardation of progression of the disease, and mayinclude the suppression of symptom recurrence in an asymptomaticpatient.

The term “anti-SAP” in relation to an “antibody”, i.e. an “anti-SAPantibody”, means an antibody that binds to human SAP with no orinsignificant binding to any other proteins, including closely relatedmolecules such as C-reactive protein (CRP).

The term “CDR” means complementarity determining region. A CDR of anantibody as used herein means a CDR as determined using any of the wellknown CDR numbering methods, including Kabat, Chothia, AbM and contactmethods.

By “circulating SAP” is meant SAP that is present in free form in theplasma in vivo and in vitro and is not associated with amyloid depositsin the tissues.

Pharmaceutical Compositions

In order to use the compound of Formula (I) in therapy, it will normallybe formulated into a pharmaceutical composition in accordance withstandard pharmaceutical practice.

The invention therefore provides a pharmaceutical composition, whichcomprises a therapeutically effective amount of a compound of Formula(I) and optionally one or more pharmaceutically acceptable carriersand/or excipients.

The present invention also provides a process for preparing apharmaceutical composition, the process comprising admixing atherapeutically effective amount of a compound of Formula (I) andoptionally one or more pharmaceutically acceptable carriers and/orexcipients.

The present invention also provides a dosage form comprising thepharmaceutical composition of the invention.

A pharmaceutical composition of the invention, which may be prepared byadmixture, suitably at ambient temperature and atmospheric pressure, isusually adapted for oral administration and, as such, may be in the formof tablets, capsules, oral liquid preparations, powders, granules, orlozenges.

In one embodiment, there is provided the pharmaceutical composition ofthe invention for oral administration.

Tablets and capsules for oral administration may be in unit dose form,and may contain conventional excipients, such as binding agents (e.g.pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g. lactose, microcrystalline cellulose orcalcium hydrogen phosphate); tabletting lubricants (e.g. magnesiumstearate, talc or silica); disintegrants (e.g. potato starch or sodiumstarch glycollate); and acceptable wetting agents (e.g. sodium laurylsulphate). The tablets may be coated according to methods well known innormal pharmaceutical practice.

Oral liquid preparations may be in the form of, for example, oilysuspension, non-aqueous solutions, emulsions, syrups or elixirs, or maybe in the form of a dry product for reconstitution with suitable aqueousor non-aqueous vehicle immediately prior to administration. Such liquidpreparations may contain conventional additives such as suspendingagents (e.g. sorbitol syrup, cellulose derivatives or hydrogenatededible fats), emulsifying agents (e.g. lecithin or acacia), non-aqueousvehicles (which may include edible oils e.g. almond oil, oily esters,ethyl alcohol or fractionated vegetable oils), preservatives (e.g.methyl or propyl-p-hydroxybenzoates or sorbic acid), and, if desired,conventional flavourings or colorants, buffer salts and sweeteningagents as appropriate. Preparations for oral administration may besuitably formulated to give controlled release of the active compound.

In another embodiment, the dosage form is a tablet or a capsule.

The composition may contain from 0.1% to 99% by weight, preferably from10 to 60% by weight, of the active material, depending on the method ofadministration. The dose of the compound used in the treatment of theaforementioned disorders will vary in the usual way with the seriousnessof the disorders, the weight of the sufferer, and other similar factors.However, as a general guide suitable unit doses may be 0.05 to 5000 mg,1.0 to 1000 mg, or 100 to 600 mg, for example 100, 200 or 300 mg, andsuch unit doses may be administered more than once a day, for exampletwo or three times a day. Such therapy may extend for a number of days,weeks, months or years.

The invention further provides the compound of Formula (I) or apharmaceutical composition as herein described, as for use in therapy.

The compound of Formula (I) is therefore of use in the treatment ofdiseases wherein SAP depletion would be beneficial.

The invention further provides the compound of Formula (I) or apharmaceutical composition as herein described, for use in depletion ofSAP.

In a further aspect there is provided a method of treatment of a diseaseor disorder in a subject wherein SAP depletion would be beneficial,which method comprises administration of a therapeutically effectiveamount of the compound of Formula (I).

In a further aspect there is provided a method of treatment of a diseaseor disorder in a subject wherein SAP depletion would be beneficial,which method comprises administration of a therapeutically effectiveamount of a pharmaceutical composition as herein described.

The invention further provides a method of depletion of SAP in asubject, which method comprises administering to the subject atherapeutically effective amount of the compound of Formula (I).

The invention further provides a method of depletion of SAP in asubject, which method comprises administering to the subject atherapeutically effective amount of a pharmaceutical composition asherein described.

Many forms of transmissible spongiform encephalopathy (prion diseases)are associated with amyloid deposits in the brain, and the presentinvention therefore relates to all these diseases or disorders,including variant Creutzfeldt-Jakob disease in humans, Creutzfeldt-Jakobdisease itself, kuru and the various other forms of human prion disease,and also bovine spongiform encephalopathy, chronic wasting disease ofmule-deer and elk, and transmissible encephalopathy of mink.

The invention further provides a method of treatment of a disease ordisorder in a subject, wherein the disease or disorder is selected fromthe group consisting of amyloidosis, Alzheimer's disease, type 2diabetes mellitus, osteoarthritis, bacterial infections, invasivecandiasis, transmissible spongiform encephalopathy, variantCreutzfeld-Jakob disease in humans, Creutzfeld-Jakob disease, kuru,other human prion diseases, bovine spongiform encephalopathy, chronicwasting disease of mule-deer and elk, and transmissible encephalopathyof mink, which method comprises administering to the subject atherapeutically effective amount of the compound of Formula (I).

The invention further provides a method of treatment of a disease ordisorder in a subject, wherein the disease or disorder is selected fromthe group consisting of amyloidosis, Alzheimer's disease, type 2diabetes mellitus, osteoarthritis, bacterial infections, invasivecandiasis, transmissible spongiform encephalopathy, variantCreutzfeld-Jakob disease in humans, Creutzfeld-Jakob disease, kuru,other human prion diseases, bovine spongiform encephalopathy, chronicwasting disease of mule-deer and elk, and transmissible encephalopathyof mink, which method comprises administering to the subject atherapeutically effective amount of a pharmaceutical composition asherein described.

The invention further provides a method of treatment of a disease ordisorder in a subject, wherein the disease or disorder is selected fromthe group consisting of amyloidosis, Alzheimer's disease, type 2diabetes mellitus and osteoarthritis, which method comprisesadministering to the subject a therapeutically effective amount of thecompound of Formula (I).

The invention further provides a method of treatment of a disease ordisorder in a subject, wherein the disease or disorder is selected fromthe group consisting of amyloidosis, Alzheimer's disease, type 2diabetes mellitus and osteoarthritis, which method comprisesadministering to the subject a therapeutically effective amount of apharmaceutical composition as herein described.

The invention further provides a method of treatment of a disease ordisorder in a subject, wherein the disease or disorder is selected fromthe group consisting of Alzheimer's disease, type 2 diabetes mellitusand osteoarthritis, which method comprises administering to the subjecta therapeutically effective amount of the compound of Formula (I).

The invention further provides a method of treatment of a disease ordisorder in a subject, wherein the disease or disorder is selected fromthe group consisting of Alzheimer's disease, type 2 diabetes mellitusand osteoarthritis, which method comprises administering to the subjecta therapeutically effective amount of a pharmaceutical composition asherein described.

The invention further provides a method of treatment of a disease ordisorder in a subject, wherein the disease or disorder is amyloidosis,which method comprises administering to the subject a therapeuticallyeffective amount of the compound of Formula (I).

The invention further provides a method of improvement of efficacy ofhuman DNA vaccines, which method comprises administering to the subjecta therapeutically effective amount of the compound of Formula (I).

The invention further provides a method of improvement of efficacy ofhuman DNA vaccines, which method comprises administering to the subjecta therapeutically effective amount of a pharmaceutical composition asherein described.

The invention further provides the use of the compound of formula (I) incombination with a DNA vaccine.

The invention further provides for the compound of Formula (I) for usein the improvement of the efficacy of human DNA vaccines.

The invention further provides for a pharmaceutical composition asherein described for use in the improvement of the efficacy of human DNAvaccines.

The invention further provides for the use of the compound of Formula(I) in the improvement of the efficacy of human DNA vaccines.

The invention further provides for the use of a pharmaceuticalcomposition as herein described in the improvement of the efficacy ofhuman DNA vaccines.

The invention further provides the use of the compound of Formula (I) inthe manufacture of a medicament for use in the improvement of theefficacy of human DNA vaccines.

By “improvement in efficacy of human DNA vaccines” is meant enabling theDNA vaccine to induce immunoprotective immune responses against theimmunogens encoded by the human DNA vaccine.

The subject may be a mammal. The subject is typically a human.

The invention further provides a method of treatment of a disease ordisorder in a subject, wherein the disease or disorder is selected fromthe group consisting of transmissible spongiform encephalopathy, variantCreutzfeldt-Jakob disease in humans, Creutzfeldt-Jakob disease, kuru,bovine spongiform encephalopathy, chronic wasting disease of mule-deerand elk, and transmissible encephalopathy of mink, which methodcomprises administering to the subject a therapeutically effectiveamount of the compound of Formula (I).

The invention further provides a method of treatment of a disease ordisorder in a subject, wherein the disease or disorder is selected fromthe group consisting transmissible spongiform encephalopathy, variantCreutzfeldt-Jakob disease in humans, Creutzfeldt-Jakob disease, kuru,bovine spongiform encephalopathy, chronic wasting disease of mule-deerand elk, and transmissible encephalopathy of mink, which methodcomprises administering to the subject a therapeutically effectiveamount of a pharmaceutical composition as herein described.

The term “amyloidosis” encompasses both systemic amyloidosis (including,but not limited to, AL-type amyloidosis, AA-type amyloidosis, dialysisamyloidosis, ATTR (transthyretin) amyloidosis, hereditary systemicamyloidosis) and local amyloidosis (including, but not limited tocerebral amyloid angiopathy).

In another aspect, the invention provides for the compound of Formula(I) or a pharmaceutical composition as herein described for use in thetreatment of a disease or disorder wherein SAP depletion would bebeneficial.

In another aspect, the invention provides for the compound of Formula(I) or a pharmaceutical composition as herein described for use in thedepletion of SAP.

In one embodiment, the invention provides for the compound of Formula(I) or a pharmaceutical composition as herein described for use in thedepletion of SAP in vivo.

In another aspect, the invention provides for the compound of Formula(I) for use in the treatment of a disease or disorder selected from thegroup consisting of amyloidosis, Alzheimer's disease, type 2 diabetesmellitus and osteoarthritis.

In another aspect, the invention provides for a pharmaceuticalcomposition as herein described for use in the treatment of a disease ordisorder selected from the group consisting of amyloidosis, Alzheimer'sdisease, type 2 diabetes mellitus and osteoarthritis.

In one embodiment, the invention provides for the compound of Formula(I) for use in the treatment of a disease or disorder selected from thegroup consisting of Alzheimer's disease, type 2 diabetes mellitus andosteoarthritis.

In one embodiment, the invention provides for the compound of Formula(I) for use in the treatment of amyloidosis.

In another aspect, the invention provides for a pharmaceuticalcomposition as herein described for use in the treatment of a disease ordisorder selected from the group consisting of Alzheimer's disease, type2 diabetes mellitus and osteoarthritis.

In another aspect, the invention provides for a pharmaceuticalcomposition as herein described for use in the treatment of amyloidosis.

In another aspect, the invention provides for the use of a compound ofFormula (I) or one or more pharmaceutical compositions herein describedin the treatment of a disease or disorder wherein SAP depletion would bebeneficial.

In another aspect, the invention provides for the use of a compound ofFormula (I) or one or more pharmaceutical compositions herein describedin the depletion of SAP.

In another aspect, the invention provides for the use of a compound ofFormula (I) in the treatment of a disease or disorder selected from thegroup consisting of amyloidosis, Alzheimer's disease, type 2 diabetesmellitus and osteoarthritis.

In another aspect, the invention provides for the use of apharmaceutical composition herein described in the treatment of adisease or disorder selected from the group consisting of amyloidosis,Alzheimer's disease, type 2 diabetes mellitus and osteoarthritis.

In another aspect, the invention provides for the use of a compound ofFormula (I) in the treatment of a disease or disorder selected from thegroup consisting of Alzheimer's disease, type 2 diabetes mellitus andosteoarthritis.

In another aspect, the invention provides for the use of a compound ofFormula (I) in the treatment of amyloidosis.

In another aspect, the invention provides for the use of apharmaceutical composition herein described in the treatment of adisease or disorder selected from the group consisting of Alzheimer'sdisease, type 2 diabetes mellitus and osteoarthritis.

In another aspect, the invention provides for the use of apharmaceutical composition herein described in the treatment ofamyloidosis.

In another aspect, the invention provides for the use of a compound ofFormula (I) in the manufacture of a medicament for use in the treatmentof a disease or disorder wherein SAP depletion would be beneficial.

In another aspect, the invention provides for the use of a compound ofFormula (I) in the manufacture of a medicament for use in the depletionof SAP.

In another aspect, the invention provides for the use of a compound ofFormula (I) in the manufacture of a medicament for use in the treatmentof a disease or disorder selected from the group consisting ofamyloidosis, Alzheimer's disease, type 2 diabetes mellitus andosteoarthritis.

In another aspect, the invention provides for the use of a compound ofFormula (I) in the manufacture of a medicament for use in the treatmentof a disease or disorder selected from the group consisting ofAlzheimer's disease, type 2 diabetes mellitus and osteoarthritis.

In another aspect, the invention provides for the use of a compound ofFormula (I) in the manufacture of a medicament for use in the treatmentof amyloidosis.

When used for the treatment of amyloidosis, the compounds of Formula (I)may be administered with an anti-SAP antibody.

In an embodiment, the anti-SAP antibody binds to the A face of humanSAP. In an embodiment, the anti-SAP antibody comprises the heavy chaincomplementarity determining regions (CDRs) present within SEQ ID NO:28(SEQ ID NO:1) and the light chain CDRs present within SEQ ID NO:35 (SEQID NO:2) in WO 11/107480. In an embodiment the anti-SAP antibodycomprises a heavy chain variable region of SEQ ID NO:28 (SEQ ID NO:1)and a light chain variable region of SEQ ID NO:35 (SEQ ID NO:2) in WO11/107480. In an embodiment, the anti-SAP antibody comprises a humanIgGI or IgG3 human constant domain. In an embodiment the anti-SAPantibody consists of a heavy chain of SEQ ID NO:62 (SEQ ID NO:3) and alight chain of SEQ ID NO:64 (SEQ ID NO:4) in WO 11/107480.

In an embodiment, the anti-SAP antibody comprises the heavy and lightchain CDRs described within WO 11/107480 as SEQ ID NO:1 (SEQ ID NO:5),SEQ ID NO:2 (SEQ ID NO:6) and SEQ ID NO:3 (SEQ ID NO:7), SEQ ID NO:4(SEQ ID NO:8), SEQ ID NO:5 (SEQ ID NO:9) and SEQ ID NO:6 (SEQ ID NO:10).

Therefore, in a further aspect, the invention provides for a method oftreatment of amyloidosis which method comprises administering to asubject a therapeutically effective amount of a compound of Formula (I)or a pharmaceutical composition as herein described in co-administrationwith an anti-SAP antibody.

In one embodiment, the administration of the compound of Formula (I) inco-administration with an anti-SAP antibody is sequential.

Such sequential administration may be close in time (eg. simultaneously)or remote in time. Furthermore, it does not matter if the compounds areadministered in the same dosage form, e.g. one compound may beadministered intravenously and the other compound may be administeredorally.

In a further embodiment, the compound of Formula (I) is administeredfirst. In a further embodiment, the anti-SAP antibody is administeredwhen the level of circulating SAP in the subject has been reduced to alevel of less than 2 mg/L. In one embodiment, the level of circulatingSAP has been reduced to a level of 1 mg/L or less. In a furtherembodiment the level of circulating SAP has been reduced to a level of0.5 mg/L or less.

The level of circulating SAP can be measured using a commerciallyavailable ELISA (enzyme-linked immunosorbent assay) kit (e.g. HK331Human SAP ELISA Kit from Hycult Biotech).

In a further embodiment, the compound of Formula (I) is administered for5-8 days or until the level of the SAP circulating in the subject hasbeen reduced to a level of less than 2 mg/L whichever is longer. In oneembodiment, the level of the SAP circulating in the subject has beenreduced to 1 mg/L or less. In a further embodiment, the level of SAPcirculating in the subject has been reduced to 0.5 mg/L or less. In afurther embodiment administration of the compound of Formula (I) iscontinued while a single dose of 200-2000 mg (preferably 250-1000 mg,more preferably 250-600 mg) of anti-SAP antibody is administered, andfor 4-6 days thereafter. This constitutes a ‘therapeuticcourse’—patients may require several courses to achieve the desiredtherapeutic effect. They are also likely to require intermittent repeattreatment. In one embodiment, the therapeutic course is repeated atleast once at 3-6 week intervals as required. In a further embodiment,the therapeutic course is repeated at least once at 3-6 week intervalsfollowed by at least one therapeutic course at 6-12 month intervals asrequired.

In a further aspect there is provided a method of treatment of a diseaseor disorder in a subject wherein SAP depletion would be beneficial,which method comprises administration of a therapeutically effectiveamount of the compound of Formula (I) in co-administration with ananti-SAP antibody.

In a further aspect there is provided a method of treatment of a diseaseor disorder in a subject wherein SAP depletion would be beneficial,which method comprises administration of a therapeutically effectiveamount of a pharmaceutical composition as herein described inco-administration with an anti-SAP antibody.

In a further aspect there is provided a method of depletion of SAP,which method comprises administration of a therapeutically effectiveamount of the compound of Formula (I) in co-administration with ananti-SAP antibody.

In a further aspect there is provided a method of depletion of SAP,which method comprises administration of a therapeutically effectiveamount of a pharmaceutical composition as herein described inco-administration with an anti-SAP antibody.

The invention further provides a method of treatment of a disease ordisorder in a subject, wherein the disease or disorder is selected fromthe group consisting of amyloidosis, Alzheimer's disease, type 2diabetes mellitus and osteoarthritis, which method comprisesadministering to the subject a therapeutically effective amount of acompound of Formula (I) in co-administration with an anti-SAP antibody.

The invention further provides a method of treatment of a disease ordisorder in a subject, wherein the disease or disorder is selected fromthe group consisting of amyloidosis, Alzheimer's disease, type 2diabetes mellitus and osteoarthritis, which method comprisesadministering to the subject a therapeutically effective amount of apharmaceutical composition as herein described in co-administration withan anti-SAP antibody.

In another aspect, the invention provides for the compound of Formula(I) or one or more pharmaceutical compositions herein described inco-administration with an anti-SAP antibody for use in the treatment ofa disease or disorder wherein SAP depletion would be beneficial.

In another aspect, the invention provides for the compound of Formula(I) or one or more pharmaceutical compositions herein described inco-administration with an anti-SAP antibody for use in the depletion ofSAP.

In another aspect, the invention provides for the compound of Formula(I) in co-administration with an anti-SAP antibody for use in thetreatment of a disease or disorder selected from the group consisting ofamyloidosis, Alzheimer's disease, type 2 diabetes mellitus andosteoarthritis.

In another aspect, the invention provides for the compound of Formula(I) in co-administration with an anti-SAP antibody for use in thetreatment of amyloidosis.

In one embodiment, the invention provides for the compound of Formula(I) in co-administration with an anti-SAP antibody for use in thetreatment of systemic amyloidosis.

In one embodiment, the invention provides for the compound of Formula(I) in co-administration with an anti-SAP antibody for use in thetreatment of AL-type amyloidosis.

In one embodiment, the invention provides for the compound of Formula(I) in co-administration with an anti-SAP antibody for use in thetreatment of AA-type amyloidosis.

In one embodiment, the invention provides for the compound of Formula(I) in co-administration with an anti-SAP antibody for use in thetreatment of dialysis amyloidosis.

In one embodiment, the invention provides for the compound of Formula(I) in co-administration with an anti-SAP antibody for use in thetreatment of ATTR (transthyretin) amyloidosis.

In one embodiment, the invention provides for the compound of Formula(I) in co-administration with an anti-SAP antibody for use in thetreatment of hereditary systemic amyloidosis.

In one embodiment, the invention provides for the compound of Formula(I) in co-administration with an anti-SAP antibody for use in thetreatment of local amyloidosis.

In one embodiment, the invention provides for the compound of Formula(I) in co-administration with an anti-SAP antibody for use in thetreatment of cerebral amyloid angiopathy.

In another aspect, the invention provides for a pharmaceuticalcomposition as herein described in co-administration with an anti-SAPantibody for use in the treatment of a disease or disorder selected fromthe group consisting of amyloidosis, Alzheimer's disease, type 2diabetes mellitus and osteoarthritis.

In another aspect, the invention provides for a pharmaceuticalcomposition as herein described in co-administration with an anti-SAPantibody for use in the treatment of amyloidosis.

In one embodiment, the invention provides for a pharmaceuticalcomposition as herein described in co-administration with an anti-SAPantibody for use in the treatment of systemic amyloidosis.

In one embodiment, the invention provides for a pharmaceuticalcomposition as herein described in co-administration with an anti-SAPantibody for use in the treatment of AL-type amyloidosis.

In one embodiment, the invention provides for a pharmaceuticalcomposition as herein described in co-administration with an anti-SAPantibody for use in the treatment of AA-type amyloidosis.

In one embodiment, the invention provides for a pharmaceuticalcomposition as herein described in co-administration with an anti-SAPantibody for use in the treatment of dialysis amyloidosis.

In one embodiment, the invention provides for a pharmaceuticalcomposition as herein described in co-administration with an anti-SAPantibody for use in the treatment of ATTR (transthyretin) amyloidosis.

In one embodiment, the invention provides for a pharmaceuticalcomposition as herein described in co-administration with an anti-SAPantibody for use in the treatment of hereditary systemic amyloidosis.

In one embodiment, the invention provides for a pharmaceuticalcomposition as herein described in co-administration with an anti-SAPantibody for use in the treatment of local amyloidosis.

In one embodiment, the invention provides for a pharmaceuticalcomposition as herein described in co-administration with an anti-SAPantibody for use in the treatment of cerebral amyloid angiopathy.

In another aspect, the invention provides for the use of a compound ofFormula (I) or a pharmaceutical composition as herein described inco-administration with an anti-SAP antibody in the treatment of adisease or disorder wherein SAP depletion would be beneficial.

In another aspect, the invention provides for the use of a compound ofFormula (I) or a pharmaceutical composition as herein described inco-administration with an anti-SAP antibody for use in the depletion ofSAP.

In another aspect, the invention provides for the use of a compound ofFormula (I) in co-administration with an anti-SAP antibody in thetreatment of a disease or disorder selected from the group consisting ofamyloidosis, Alzheimer's disease, type 2 diabetes mellitus andosteoarthritis.

In another aspect, the invention provides for the use of a compound ofFormula (I) in co-administration with an anti-SAP antibody in thetreatment of amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) in co-administration with an anti-SAP antibody in thetreatment of systemic amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) in co-administration with an anti-SAP antibody in thetreatment of AL-type amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) in co-administration with an anti-SAP antibody in thetreatment of AA-type amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) in co-administration with an anti-SAP antibody in thetreatment of dialysis amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) in co-administration with an anti-SAP antibody in thetreatment of ATTR (transthyretin) amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) in co-administration with an anti-SAP antibody in thetreatment of hereditary systemic amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) in co-administration with an anti-SAP antibody in thetreatment of local amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) in co-administration with an anti-SAP antibody in thetreatment of cerebral amyloid angiopathy.

In another aspect, the invention provides for the use of apharmaceutical composition as herein described in co-administration withan anti-SAP antibody in the treatment of a disease or disorder selectedfrom the group consisting of amyloidosis, Alzheimer's disease, type 2diabetes mellitus and osteoarthritis.

In another aspect, the invention provides for the use of apharmaceutical composition as herein described in co-administration withan anti-SAP antibody in the treatment of amyloidosis.

In one embodiment, the invention provides for the use of apharmaceutical composition as herein described in co-administration withan anti-SAP antibody in the treatment of systemic amyloidosis.

In one embodiment, the invention provides for the use of apharmaceutical composition as herein described in co-administration withan anti-SAP antibody in the treatment of AL-type amyloidosis.

In one embodiment, the invention provides for the use of apharmaceutical composition as herein described in co-administration withan anti-SAP antibody in the treatment of AA-type amyloidosis.

In one embodiment, the invention provides for the use of apharmaceutical composition as herein described in co-administration withan anti-SAP antibody in the treatment of dialysis amyloidosis.

In one embodiment, the invention provides for the use of apharmaceutical composition as herein described in co-administration withan anti-SAP antibody in the treatment of ATTR (transthyretin)amyloidosis.

In one embodiment, the invention provides for the use of apharmaceutical composition as herein described in co-administration withan anti-SAP antibody in the treatment of hereditary systemicamyloidosis.

In one embodiment, the invention provides for the use of apharmaceutical composition as herein described in co-administration withan anti-SAP antibody in the treatment of local amyloidosis.

In one embodiment, the invention provides for the use of apharmaceutical composition as herein described in co-administration withan anti-SAP antibody in the treatment of cerebral amyloid angiopathy.

In another aspect, the invention provides for the use of a compound ofFormula (I) and an anti-SAP antibody in the manufacture of a medicamentfor use in the treatment of a disease or disorder wherein SAP depletionwould be beneficial.

In another aspect, the invention provides for the use of a compound ofFormula (I) and an anti-SAP antibody in the manufacture of a medicamentfor use in the depletion of SAP.

In another aspect, the invention provides for the use of a compound ofFormula (I) and an anti-SAP antibody in the manufacture of a medicamentfor use in the treatment of a disease or disorder selected from thegroup consisting of amyloidosis, Alzheimer's disease, type 2 diabetesmellitus and osteoarthritis.

In another aspect, the invention provides for the use of a compound ofFormula (I) and an anti-SAP antibody in the manufacture of a medicamentfor use in the treatment of amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) and an anti-SAP antibody in the manufacture of a medicamentfor use in the treatment of systemic amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) and an anti-SAP antibody in the manufacture of a medicamentfor use in the treatment of AL-type amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) and an anti-SAP antibody in the manufacture of a medicamentfor use in the treatment of AA-type amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) and an anti-SAP antibody in the manufacture of a medicamentfor use in the treatment of dialysis amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) and an anti-SAP antibody in the manufacture of a medicamentfor use in the treatment of ATTR (transthyretin) amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) and an anti-SAP antibody in the manufacture of a medicamentfor use in the treatment of hereditary systemic amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) and an anti-SAP antibody in the manufacture of a medicamentfor use in the treatment of local amyloidosis.

In one embodiment, the invention provides for the use of a compound ofFormula (I) and an anti-SAP antibody in the manufacture of a medicamentfor use in the treatment of cerebral amyloid angiopathy.

In one embodiment the disease or disorder is amyloidosis.

In a further embodiment the disease or disorder is systemic amyloidosis.

In a further embodiment the disease or disorder is AL-type amyloidosis.

In a further embodiment the disease or disorder is AA-type amyloidosis.

In a further embodiment the disease or disorder is dialysis amyloidosis.

In a further embodiment the disease or disorder is ATTR (transthyretin)amyloidosis.

In a further embodiment the disease or disorder is hereditary systemicamyloidosis.

In another embodiment the disease or disorder is local amyloidosis.

In a further embodiment the disease or disorder is cerebral amyloidangiopathy.

In one embodiment the disease or disorder is Alzheimer's disease.

In one embodiment the disease or disorder is type 2 diabetes mellitus.

In one embodiment the disease or disorder is osteoarthritis.

In another embodiment of the invention, an article of manufacture, or“kit of parts”, containing one or more unit doses of an anti-SAPantibody and one or more unit doses of the compound of Formula (I),useful for the treatment of amyloidosis, is provided.

In one embodiment the kit of parts comprises a unit dose of an anti-SAPantibody and one or more unit doses of the compound of Formula (I).

Suitably the kit of parts is formulated for the separate or sequentialadministration of the one or more unit doses of compound of Formula (I)and the unit dose of the anti-SAP antibody.

In one embodiment, the kit of parts comprises a container comprising oneor more unit doses of the compound of Formula (I) or a pharmaceuticalcomposition as herein described and the unit dose of the anti-SAPantibody.

In another embodiment, the kit of parts comprises a first containercomprising one or more unit doses of the compound of Formula (I) or apharmaceutical composition as herein described and a second containercomprising the unit dose of the anti-SAP antibody.

The kit of parts may further comprise directions for the administrationof the one or more unit doses of compound of Formula (I) and the unitdose of the anti-SAP antibody for treating or preventing amyloidosis.

In an alternative embodiment of the invention, an article ofmanufacture, or “kit of parts”, containing one or more unit doses of thecompound of Formula (I) or a pharmaceutical composition as hereindescribed and one or more unit doses of a DNA vaccine is provided.

Suitable containers include, for example, bottles, vials, syringes andblister packs, etc.

WO2011/139917 discloses anti-transthyretin (anti-TTR) antisenseoligonucleotides potentially useful in the modulation of expression oftransthyretin and in treating, preventing, delaying or amelioratingtransthyretin amyloidosis.

In another aspect, the invention provides for a method of treatment ofATTR (transthyretin) amyloidosis, which method comprises i)administering to a subject a therapeutically effective amount of acompound of formula (I) or a pharmaceutical composition as hereindescribed in co-administration with an anti-SAP antibody, and ii)administering to a subject a therapeutically effective amount of ananti-TTR antisense oligonucleotide.

In one embodiment of the invention, the anti-TTR antisenseoligonucleotide is ISIS 420915.

In one embodiment, steps i) and ii) are carried out sequentially.

In one embodiment, step ii) is carried out after step i).

WO2009040405 discloses agents for stabilising the tetrameric form oftransthyretin useful in the treatment or prevention of transthyretinamyloidosis.

Therefore, in another aspect, the invention provides for a method oftreatment of ATTR (transthyretin) amyloidosis, which method comprises i)administering to a subject a therapeutically effective amount of acompound of formula (I) or a pharmaceutical composition as hereindescribed in co-administration with an anti-SAP antibody, and ii)administering to a subject a therapeutically effective amount of anagent as described in WO2009040405.

In one embodiment, steps i) and ii) are carried out sequentially.

In one embodiment, step ii) is carried out after step i).

The compound of Formula (I) may be synthesised substantially accordingto Reaction Scheme 1.

The compounds of Formula (I) can be prepared by reaction of thechloromethyl carbonate of Formula (II) with CPHPC in the presence of asolvent (e.g. dioxane), a base (e.g. potassium carbonate) and catalyticamounts of TBAI (tetrabutylammonium iodide).

The chloromethyl carbonate of Formula (II) can be prepared by reactionof chloromethyl carbonochloridate with tetrahydro-2H-pyran-4-ol in thepresence of a solvent (e.g. diethyl ether or dichloromethane) and a base(e.g. pyridine or dimethylaminopyridine).

Chloromethyl carbonochloridate (also known as chloromethylchloroformate) and tetrahydro-2H-pyran-4-ol are commercially available(Aldrich).

Alternatively, the compounds of Formula (I) can be synthesisedsubstantially according to Reaction Scheme 2 starting from D-proline((R)-pyrrolidine-2-carboxylic acid).

The compound in the box is CPHPC.

D-proline is commercially available (Aldrich).

EXAMPLES

The following Example illustrates the invention. This Example is notintended to limit the scope of the present invention, but rather toprovide guidance to the skilled artisan to prepare and use the compound,compositions, and methods of the present invention. While particularembodiments of the present invention are described, the skilled artisanwill appreciate that various changes and modifications can be madewithout departing from the spirit and scope of the invention.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The following Intermediates and Examples illustrate the preparation ofthe compound of the invention.

Abbreviations

-   2-MeTHF 2-methyltetrahydrofuran-   aq. aqueous-   BnOH benzyl alcohol-   Bu₄NI tetrabutylammonium iodide-   DCM dichloromethane/methylene chloride-   DMAP dimethylaminopyridine-   ESI electrospray ionisation-   EtOAc ethyl acetate-   Et₂O diethyl ether-   h hour-   HCl hydrogen chloride/hydrochloric acid-   HPLC high performance liquid chromatography-   IPA isopropyl alcohol-   ^(i)Pr₂O isopropyl ether-   MeCN/CH₃CN acetonitrile-   Min minute-   MS mass spectrometry-   Na₂SO₄ sodium sulphate-   NMR nuclear magnetic resonance-   PhMe toluene-   py pyridine-   RT room temperature-   TBAI tetrabutylammonium iodide-   TEA triethylamine-   TOF time of flight-   THF tetrahydrofuran

¹H and ¹³C NMR spectra were recorded on a Bruker 300 or 400 MHz.Chemical shifts are reported in parts per million (ppm, units).High-resolution mass spectra were recorded on a Micromass LCT (TOF)spectrometer coupled to analytical high performance liquidchromatography (HPLC). HPLC was conducted on a Waters X-Terra MS C18column (3.5 μm 30×4.6 mm id) eluting with 0.01M ammonium acetate inwater (solvent A) and 100% acetonitrile (solvent B), using the followingelution gradient 0-0.5 minutes 5% B, 0.5-3.75 minutes 5% to 100% B,3.75-4.5 100% B, 4.5-5 100% to 5% B, 5-5.5 5% B at a flow rate of 1.3ml/minute at 40° C. The mass spectra (MS) were recorded on a Waters LCTmass spectrometer using electrospray positive ionisation [ES⁺ve to giveMH⁺ molecular ions] or electrospray negative ionisation [ES-ve to give(M−H)⁻ molecular ions] modes.

Analytical HPLC was conducted on a XSelect XP C18 column (2.5 μm 30×4.6mm id) eluting with 0.1% formic acid in water (solvent A) and 0.1%formic acid in acetonitrile (solvent B), using the following elutiongradient 0-3.2 minutes: 5% to 100% B, 3.2-4.0 minutes 100% B, at a flowrate of 1.8 ml/minute at 40° C. The mass spectra (MS) were recorded on aWaters ZQ mass spectrometer using electrospray positive ionisation [ES⁺to give MH⁺ molecular ions] or electrospray negative ionisation [ES⁻ togive (M−H)⁻ molecular ions] modes.

Intermediate 1: Chloromethyl (tetrahydro-2H-pyran-4-yl) carbonate

To a solution of tetrahydro-2H-pyran-4-ol (40 g, 392 mmol) in diethylether (500 mL) was added pyridine (38.0 mL, 470 mmol) and the solutionwas cooled to 0° C. Chloromethyl carbonochloridate (41.8 mL, 470 mmol)was added dropwise leading to the formation of a white solid. Thereaction mixture was stirred at RT for 18 h. The reaction mixture waswashed with water (200 mL), with HCl 0.5N (200 mL), and then with asaturated solution of NaHCO₃ (200 mL). The organic phase was dried overNa₂SO₄ and concentrated under reduced pressure.

Toluene was added and the solution was concentrated under reducedpressure (to remove chloromethyl carbonochloridate in excess).Chloromethyl (tetrahydro-2H-pyran-4-yl) carbonate (Intermediate 1) wasobtained as a colourless oil (70 g, 360 mmol, 92% yield).

¹H NMR (400 MHz, CDCl₃) δ ppm 5.75 (s, 2H), 4.92 (m, 1H), 3.95 (m, 2H),3.56 (m, 2H), 2.02 (m, 2H), 1.80 (m, 2H).

Intermediate 1 (Alternative preparation): Chloromethyl(tetrahydro-2H-pyran-4-yl) carbonate

To a solution of chloromethyl carbonochloridate (5 g, 38.8 mmol) in DCM(50 mL) was added tetrahydro-2H-pyran-4-ol (3.96 g, 38.8 mmol) and thesolution was cooled to 0° C. DMAP (4.97 g, 40.7 mmol) was added then thereaction mixture was stirred at RT for 18 h. The reaction mixture wasdiluted with water and extracted with DCM (3×100 mL). The organic phaseswere combined, dried over Na₂SO₄ and concentrated under reducedpressure. The pale yellow oil was purified by chromatography elutingwith 10% EtOAc in cyclohexane. The appropriate fractions were combinedand concentrated in vacuo to give the required product as a colorlessoil (2.2 g, 11.3 mmol, 29.2% yield).

¹H NMR (300 MHz, CDCl₃) δ ppm 5.76 (s, 2H), 4.92 (m, 1H), 3.94 (m, 2H),3.57 (m, 2H), 2.02 (m, 2H), 1.80 (m, 2H).

Example 1(2R,2′R)-Bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl)1,1′-adipoylbis(pyrrolidine-2-carboxylate)

Solution A: Potassium carbonate (29.8 g, 216 mmol) was added to astirred suspension of (2R,2′R)-1,1′-adipoylbis(pyrrolidine-2-carboxylicacid) (35 g, 103 mmol) in 1,4-dioxane (1 L) and the reaction mixture wasstirred at 80° C. for 30 min.

Solution B: TBAI (7.60 g, 20.57 mmol) was added to a solution ofchloromethyl (tetrahydro-2H-pyran-4-yl) carbonate (42.0 g, 216 mmol) indioxane (50 mL) and the mixture was stirred at RT for 15 min.

The solution B was added to the solution A. The reaction mixture wasstirred at 80° C. for 18 h.

The reaction mixture was filtered and concentrated under reducedpressure. The residue was taken up in EtOAc (400 mL) and washed with anaq. solution of NaHCO₃ (2×100 mL), an aq. solution of sodium thiosulfate(50 mL) and with 0.5N HCl (100 mL). The organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo. The yellow oil wassolubilized in 2-MeTHF (100 mL) and sonicated until crystallizationoccurred. The mixture was left to stand for 1 h at RT. The precipitatewas filtered and washed with a mixture 2-MeTHF/iPr₂O 70/30 to afford(2R,2′R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl)1,1′-adipoylbis(pyrrolidine-2-carboxylate) (Example 1) as an off whitepowder (42 g, 64.0 mmol, 62.2% yield). The product was dried underreduced pressure (5 mbar) and 35° C. for 12 h.

¹H NMR (400 MHz, CDCl₃) δ ppm 5.88 (d, J=5.5 Hz, 2H), 5.73 (d, J=5.5 Hz,2H), 4.87 (m, 2H), 4.50 (m, 2H), 3.93 (m, 4H), 3.65 (m, 2H), 3.55 (m,6H), 2.42-1.90 (m, 16H), 1.84-1.60 (m, 8H).

Some minor peaks were observed due to the presence of rotamers.

Recrystallisation of(2R,2′R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl)1,1-adipoylbis(pyrrolidine-2-carboxylate)

(2R,2′R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl)1,1′-adipoylbis(pyrrolidine-2-carboxylate) (170 g, 259 mmol) wassuspended in 2-MeTHF and heated to 90° C. until complete dissolution.The solution was filtered when still hot and allowed to cool to RT. Theprecipitate was filtered and washed with a mixture of 2-MeTHF/iPr₂O70/30 to afford(2R,2′R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl)1,1′-adipoylbis(pyrrolidine-2-carboxylate) (130 g, 198 mmol, 76% yield)as an off white crystalline solid. The product was dried under reducedpressure (5 mbar) and 35° C. for 24 h.

LC/MS: m/z 657 [M+H]⁺, Rt 1.98 min.

¹H NMR (400 MHz, CDCl₃) δ ppm 5.87 (d, J=5.5 Hz, 2H), 5.71 (d, J=5.5 Hz,2H), 4.86 (m, 2H), 4.49 (m, 2H), 3.91 (m, 4H), 3.63 (m, 2H), 3.54 (m,6H), 2.43-1.85 (m, 16H), 1.84-1.59 (m, 8H).

Some minor peaks were observed due to the presence of rotamers.

¹³C NMR (100 MHz, CDCl₃) 171.69, 170.85, 153.12, 82.26, 77.36, 77.05,76.72, 73.94, 65.02, 58.41, 46.95, 34.11, 31.47, 29.02, 24.80, 24.17.

HRMS: m/z calculated for C₃₀H₄₅N₂O₁₄ [M+H]⁺ 657.2870, found 657.2883.

XRPD data were acquired on a PANalytical X'Pert Pro powderdiffractometer, model PW3040/60 using an X'Celerator detector. Theacquisition conditions were: radiation: Cu Kα, generator tension: 40 kV,generator current: 45 mA, start angle: 2.0° 2θ, end angle: 40.0° 2θ,step size: 0.0167° 2θ, time per step: 31.75 seconds. The sample wasprepared by mounting a few milligrams of sample (compound of Formula(I)) on a silicon wafer (zero background plate), resulting in a thinlayer of powder.

The XRPD spectrum of the crystalline solid form of compound of Formula(I) is presented in FIG. 1. Characteristic XRPD angles and d-spacingsfor the compound of Formula (I) are recorded in Table 1. The margin oferror is approximately ±0.1° 2θ for each of the peak assignments. Peakintensities may vary from sample to sample due to preferred orientation.

Peak positions were measured using Highscore software.

TABLE 1 XRPD diffraction angles and d-spacings for compound of Formula(I) Compound of formula (I) 2θ/° d-spacings/Å 3.6 24.4 7.2 12.3 10.8 8.214.4 6.2 16.1 5.5 17.1 5.2 18.0 4.9 18.5 4.8 19.8 4.5 21.7 4.1 22.9 3.924.3 3.7 27.1 3.3

In a further aspect, the invention provides for a compound of Formula(I) in crystalline form.

In a further aspect, the invention provides for a compound of Formula(I) as a crystalline solid characterised by an XRPD spectrum that issubstantially as shown in FIG. 1.

In a further aspect, the invention provides for a compound of Formula(I) as a crystalline solid characterised by an XRPD spectrum comprisingthe peaks of Table 1.

Comparator Compounds

The data hereinafter reported compares the compound of Formula (I) with(2R,2′R)-bis(1-(pivaloyloxy)ethyl)1,1′-adipoylbis(pyrrolidine-2-carboxylate) (Comparator Compound).

(2R,2′R)-bis(1-(pivaloyloxy)ethyl)1,1′-adipoylbis(pyrrolidine-2-carboxylate) (also known as(R)-1-(6-{(R)-2-[1-(2,2-dimethyl-propionyloxy)-ethoxycarbonyl]-pyrrolidin-1-yl}-6-oxo-hexanoyl)-pyrrolidine-2-carboxylicacid 1-(2,2-dimethyl-propionyloxy)-ethyl ester) can be synthesisedaccording to the experimental protocol disclosed in WO2003/051836.

Physicochemical Properties Physical Form

The compound of Formula (I) is obtainable as a crystalline solid. Bycontrast, Comparator Compound is obtained as an oil (see WO2003/051836).

Solubility Protocol for Determining Solubility

A known quantity of the compound of Formula (I) was weighed into asuitable vessel (e.g. a screw capped clear glass vial) and a knownvolume of the required media added (e.g. Simulated Gastric Fluid pH1.6[SGF], Simulated Fed State Intestinal Fluid pH 6.5 [FeSSIF],Simulated Fasted State Intestinal Fluid pH 6.5 [FaSSIF], Water [PurifiedWater], or Britton-Robinson buffer). The compound was wetted with themedia by vortex mixing for 30 seconds to 1 minute. The sample was thenvisually observed to ensure that undissolved solid remained present. Thesample was transferred to a gentle mixer (such as a roller mixer) andallowed to agitate until the desired time point was reached. Atappropriate times the sample was reassessed visually. If all the solidhad dissolved the solubility was recorded as >x mg/ml where x is theknown weight used divided by the volume added. If undissolved solidremained a portion of the sample was taken and centrifuged to remove thesolid leaving a clear supernatant. The supernatant was dilutedvolumetrically with a suitable diluent to provide an analytical sampleof suitable concentration for analysis. This diluted sample was thenanalysed by a suitable method such as HPLC against a standard(s) ofknown concentration. The solubility of the compound can then becalculated using a knowledge of the concentration of the standard, therelative response (e.g. peak areas) of the standard and the analyticalsample described, and the dilution of the analytical sample.

The solubility of the compound of Formula (I) in various aqueous mediaare shown below in Table 2.

TABLE 2 Solubility of compound of Formula (I) in water, FeSSIF, FaSSIFand SGF at 4 hour timepoint Media Tested Solubility (mg/ml) at 4 hourtimepoint SGF 5.2 FaSSIF 4.4 FeSSIF 5.0 Water 5.5

Therefore the compound of Formula (I) is highly soluble in biologicallyrelevant media.

Cytochrome p450 and Drug-Drug Interactions

The compound of Formula (I) and Comparator Compound were assessed in thecytochrome p450 (CYP 450) assay below. The results are shown below inTable 3.

The assay was designed to evaluate inhibition on cytochrome P450 (CYP)3A4, 2C9, 2C19, 1A2 and 2D6 enzymes from bactosomes source usingfluorogenic substrates. Compound (compound of Formula (I) or ComparatorCompound) was dissolved in methanol at 1.65 mM. Daughter solutions wereprepared in methanol at 660, 264, 106, 42, 17, 6.8, 2.7, 1.1 and 0.43μM. NADPH cofactor was prepared with Glucose-6-phosphate (7.8 mg),Glucose-6-phosphate dehydrogenase (6 units), NADP (1.7 mg) per 1 mL inNaHCO₃ 2%.

Substrate preparation was as follows:

-   -   7-Methoxy-4-triFluoromethyl Coumarin-3-Acetic acid ethyl ester        (FCA): 12.5 mM in acetonitrile    -   EthoxyResorufin (ER): 0.05 mM in acetonitrile    -   4-MethylaminoMethyl-7-Methoxy Coumarin (MMC): 2.5 mM in methanol    -   3-Butyryl-7Methoxy Coumarin (BMC): 2.5 mM in DMSO    -   7-BenzyloxyQuinoline (7BQ): 2.5 mM in acetonitrile    -   DiEthoxyFluorescein (DEF): 0.1 mM in acetonitrile

Bactosomes (Cypex source) at the concentration of 10 mg of protein perml were diluted in phosphate buffer 50 mM pH7.4:

-   -   0.33 ml of bactosomes CYP2C9 with 23.8 ml of buffer and 0.11 ml        of FCA    -   0.33 ml of bactosomes CYP1A2 with 23.6 ml of buffer and 0.275 ml        of ER    -   0.33 ml of bactosomes CYP2D6 with 23.8 ml of buffer and 0.11 ml        of MMC    -   0.33 ml of bactosomes CYP2C19 with 23.8 ml of buffer and 0.11 ml        of BMC    -   0.33 ml of bactosomes CYP3A4H with 23.6 ml of buffer and 0.275        ml of 7BQ    -   0.33 ml of bactosomes CYP3A4L with 23.6 ml of buffer and 0.275        ml of DEF

Pre-Incubation consisted in mixing 5 μl of compound solution with 220 μldiluted bactosomes and warming it at 37° C. for 10 min. Incubation wasstarted with the addition of 25 μl of NADPH. Then fluorescence ofsubstrate metabolite was read in a SAFIRE instrument (from Tecan) for 5min:

-   -   FCA (2C9): Excitation at 410 nm and Emission at 510 nm    -   ER (1A2): Excitation at 530 nm and Emission at 590 nm    -   MMC (2D6): Excitation at 410 nm and Emission at 485 nm    -   BMC (2C19): Excitation at 410 nm and Emission at 465 nm    -   7BQ (3A4H): Excitation at 410 nm and Emission at 530 nm    -   DEF (3A4L): Excitation at 485 nm and Emission at 530 nm

Plotting of inhibition of substrate production against compoundconcentration allowed the determination of IC₅₀ values.

TABLE 3 Inhibition of cytochrome p450 enzymes by compound of Formula (I)(Example 1) and Comparator Compound. Compound of Comparator Formula (I)Compound CYP450 enzyme IC₅₀ (μM) IC₅₀ (μM) CYP1A2 >33 >33CYP2C19 >33 >33 CYP2C9 >33 26 CYP2D6 >33 >33 CYP3A4 (7BQ) >33 5.5 CYP3A4(DEF) >33 1.1

In fluorescence based screening assays using recombinant human CYP 450,the compound of Formula (I) and its mono-ester derivative did notdemonstrate significant inhibition of the major human liver cytochromeP450s (Cypex): CYP3A4-DEF and 3A4-7BQ with IC₅₀>33 μM. The otherisoforms were also not significantly inhibited by both compounds withIC₅₀>33 μM). By contrast, Comparator Compound demonstrated significantinhibition of CYP3A4-DEF and CYP3A4-7BQ.

For a compound where the systemic concentration is going to be low, onlyCYP3A4 inhibition is relevant as only intestinal inhibition will berelevant (CYP3A is present in enterocytes and is responsible forenterocyte first pass).

Physical Stability Protocol for Determining Physical Stability:

The assay was designed for determining physical stability of compound inbuffer at various pHs. Compound of Formula (I) was dissolved in DMSO at1 mg/ml. Phosphate buffer (PBS) was prepared by mixing K₂HPO₄ 50 mM andKH₂PO₄ 50 mM solutions to obtain 4 buffers at pH 6.0, 7.0, 7.5, 8.0.

Stability studies were conducted at room temperature, and were startedby the addition of 8 μl of DMSO solution to 792 μl of PBS 50 mM at pH6.0, 7.0, 7.5 or 8.0 (1% DMSO). 75 μl of mixture was taken at 0, 1, 2, 4and 24 h and 225 μl acetonitrile containing internal standard was added.

2 μl of samples were injected into the liquid chromatography system andeluted with a Ascentis C18 column (50×2.1 mm id, 2.7 μm) and with 0.1%formic acid in water (A) and 0.1% formic acid in acetonitrile (B), usingthe following elution 2 min gradient: 5 to 95% B over 1.2 min, 95% Bover 0.6 min and 0.1 min for re-equilibrate column, at 0.5 mL/min at 50°C.

Samples were analysed by Mass Spectrometry with an electrospray sourceand in positive mode and with following mass transitions:

-   -   Compound of Formula (I): 657 to 384    -   Monoester of CPHPC: 499 to 226    -   CPHPC: 341 to 226

Data showing in vitro physical hydrolysis of the compound of Formula (I)in phosphate buffered saline at pH 6, 7, 7.5 and 8 at various timepoints is presented in FIG. 2.

By “monoester of CPHPC” is meant the compound(R)-1-(6-oxo-6-((R)-2-((((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methoxy)carbonyl)pyrrolidin-1-yl)hexanoyl)pyrrolidine-2-carboxylicacid of Formula (III).

The compound of Formula (I) seemed to be less sensitive to hydrolysisfor a pH below 7.5. Less than 20% of the compound of Formula (I) washydrolysed after 24 h in an acidic environment (pH less than 7.0).

Intestinal and Liver Microsomal Hydrolysis Intestine and LiverMicrosomal Assay Protocol

The assay was designed for determining stability of compound inmicrosomal matrix. Compound (Compound of Formula (I)) was dissolved inDMSO at 1 mg/ml. Daughter solution was prepared in methanol/water(50/50) at 30.3 ng/ml. Microsomes (from Xenotech) were diluted at 0.625mg proteins per ml in phosphate buffer 50 mM pH 7.4.

Pre-Incubation consisted of warming microsomal solution 395 μl with 100μl NaHCO₃ (2%) at 37° C. for 7 min. Incubation was started with theaddition of 5 μl of daughter solution. 50 μl aliquots of mixture weretaken at 0, 3, 6, 12 and 30 min and quenched with 150 μl acetonitrilecontaining internal standard.

After 10 min centrifugation at 4000 rpm, 2 μl of samples were injectedinto the liquid chromatography system and eluted on a Ascentis C18column (50×2.1 mm id, 2.7 μm) with 0.1% formic acid in water (A) and0.1% formic acid in acetonitrile (B), using the following 2 minuteelution gradient: 5 to 95% B over 1.2 min, 95% B over 0.6 min and 0.1min for re-equilibrate column, at 0.5 mL/min at 50° C.

Samples were analysed by Mass Spectrometry with an electrospray source,in positive mode and with following mass transitions:

-   -   Compound of Formula (I): 657 to 384    -   Monoester of CPHPC: 499 to 226    -   CPHPC: 341 to 226

Controls were made to calculate percentage of disappearance of parentbut also appearance of suspected metabolite, i.e. monoester and diacidicform.

Data showing in vitro Intestinal Microsomal Stability of Compound ofFormula (I) in human microsomes is presented in FIG. 3. “0/5/10/30/60min” refers to the time (in minutes) that the sample was taken from themixture for analysis.

As can be seen from FIG. 3, the compound of Formula (I) exhibited a lowrate of hydrolysis in human intestinal microsomes, even after 60minutes, suggesting that the compound of Formula (I) will not be undulyunstable in the gut and so be available for absorption.

From the intestine, the compound of Formula (I) is transported throughthe intestinal wall and is transported to the bloodstream and liver,both sites of circulating SAP.

Protocol for Determining Blood Hydrolysis

The assay was designed for determining stability of compound in freshblood. Compound (compound of Formula (I)) was dissolved in DMSO at 1mg/ml. Daughter solution was prepared in DMSO at 100 μg/ml. Fresh bloodwas diluted 1/2 in isotonic buffer pH 7.4.

Pre-Incubation consisted of warming 792 μl of blood at 37° C. for 7 min.Incubations were started with the addition of 5 μl of daughter solution.50 μl of mixture were taken at 0, 5, 15, 30 and 60 min. 50 μl of waterwas add to sample and then quenched with 300 μl acetonitrile containinginternal standard.

After 10 min centrifugation at 4000 rpm, 2 μl of samples were injectedinto the liquid chromatography system and eluted with a Ascentis C18column (50×2.1 mm id, 2.7 μm) and with 0.1% formic acid in water (A) and0.1% formic acid in acetonitrile (B), using the following elutiongradient over 2 minutes: 5 to 95% B over 1.2 min, 95% B over 0.6 min and0.1 min for re-equilibrate column, at 0.5 mL/min at 50° C. Samples wereanalysed by Mass Spectrometry with an electrospray source, in positivemode and with following mass transitions:

-   -   Compound of Formula (I): 657 to 384    -   Monoester of CPHPC: 499 to 226    -   CPHPC: 341 to 226

Controls were made to calculate percentage of disappearance of parentbut also apparition of suspected metabolite, i.e. monoester and diacidicform.

Data showing in vitro Blood stability of compound of Formula (I) inhuman blood is presented in FIG. 4. “0/5/10/30/60 min” refers to thetime (in minutes) that the sample was taken from the mixture foranalysis.

In vitro liver microsomal activity of was assessed using the protocoldetailed above (Intestine and Liver Microsomal Assay protocol).

Data showing in vitro Liver microsomal stability of compound of Formula(I) in human liver microsomes is presented in FIG. 5. “0/5/10/30/60 min”refers to the time (in minutes) that the sample was taken from themixture for analysis.

In human liver microsomes, a high rate of hydrolysis of compound ofFormula (I) to CPHPC was observed (approximately 50% conversion to CPHPCwas achieved within 30 minutes), suggesting that compound of Formula (I)is capable of being cleaved to active CPHPC once absorbed.

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1. A kit of parts comprising at least one dosage form of a compoundwhich is(2R,2′R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl)1,1′-adipoylbis(pyrrolidine-2-carboxylate) according to Formula (I):

and at least one dosage form of an anti-SAP antibody wherein the aminoacid sequence of the anti-SAP antibody comprises the heavy and lightchain CDRs described within International Patent Application PublicationWO 11/107480 as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5 and SEQ ID NO:6.
 2. A method of treatment of a disease ordisorder in a subject wherein SAP depletion would be beneficial, whichmethod comprises administration of a therapeutically effective amount ofa compound which is(2R,2′R)-bis(((((tetrahydro-2H-pyran-4-yl)oxy)carbonyl)oxy)methyl)1,1′-adipoylbis(pyrrolidine-2-carboxylate) according to Formula (I):

and an anti-SAP antibody wherein the amino acid sequence of the anti-SAPantibody comprises the heavy and light chain CDRs described withinInternational Patent Application Publication WO 11/107480 as SEQ IDNO:1, SEQ ID NO:2 and SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ IDNO:6.
 3. The method according to claim 2 wherein the disease or disorderis selected from the group consisting of amyloidosis, Alzheimer'sdisease, type 2 diabetes mellitus, and osteoarthritis.
 4. The methodaccording to claim 2 wherein the disease or disorder is amyloidosis. 5.The method according to claim 2 wherein the disease or disorder issystemic amyloidosis.
 6. The method according to claim 2 wherein thecompound is co-administered with the anti-SAP antibody.
 7. The methodaccording to claim 6 wherein the co-administration is sequential.
 8. Themethod according to claim 7 wherein the compound is administered priorto administration of the anti-SAP antibody.
 9. The method according toclaim 8 wherein the anti-SAP antibody is administered when the level ofcirculating SAP in the subject has been reduced to a level of less than2 mg/L.
 10. The method according to claim 8 wherein the anti-SAPantibody is administered when the level of circulating SAP in thesubject has been reduced to a level of 1 mg/L or less.
 11. The methodaccording to claim 8 wherein the anti-SAP antibody is administered whenthe level of circulating SAP in the subject has been reduced to a levelof 0.5 mg/L or less.
 12. The method according to claim 8 wherein thecompound is administered for 5-8 days or until the level of the SAPcirculating in the subject has been reduced to a level of less than 2mg/L whichever is longer.
 13. The method according to claim 4 whereinthe compound is co-administered with the anti-SAP antibody.
 14. Themethod according to claim 13 wherein the co-administration issequential.
 15. The method according to claim 14 wherein the compound isadministered prior to administration of the anti-SAP antibody.
 16. Themethod according to claim 15 wherein the anti-SAP antibody isadministered when the level of circulating SAP in the subject has beenreduced to a level of less than 2 mg/L.
 17. The method according toclaim 15 wherein the anti-SAP antibody is administered when the level ofcirculating SAP in the subject has been reduced to a level of 1 mg/L orless.
 18. The method according to claim 15 wherein the anti-SAP antibodyis administered when the level of circulating SAP in the subject hasbeen reduced to a level of 0.5 mg/L or less.
 19. The method according toclaim 15 wherein the compound is administered for 5-8 days or until thelevel of the SAP circulating in the subject has been reduced to a levelof less than 2 mg/L whichever is longer.